This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of these polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of the relA/spoT gene family, hereinafter referred to as xe2x80x9cspo-relxe2x80x9d.
The Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. Staphylococcus aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
This invention provides novel spo-rel, which can be used, among other things, for screening for pharmaceutical compounds to treat Staphylococcal infections. In E. coli relA and spoT proteins are involved in the stringent response to nutrient limitation and regulate the accumulation of (p)ppGpp which is involved in the regulation of gene expression and other cellular processes (see Cashel, M. et al. The Stringent Response. In F. C. Neidhardt et al. (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2n edition, ASM Press, Washington, D.C., 1996). The spoT protein is capable of both the synthesis and degradation of (p)ppGpp while the relA protein is capable of (p)ppGpp synthesis only. The spoT and relA proteins are homologous being 55.6% similar and 31.9% identical (Metzger et al., J. Biol. Chem. 264:9122-9125, 1989).
The genetic organization of the streptokinase region of the Streptococcus equisimilis H46A chromosome has been defined including the sequence of a full length ORF of a relA/spoT homolog, desingated rel (Mechold, U. et al. Mol. Gen. Genet. 241:129-140, 1993). Functional analysis of the rel gene from Streptococcus equisimilis indicates that the encoded protein is similar to E. coli spoT in that it catalyzes both the synthesis and degradation of (p)ppGpp (Mechold, U., et al., J. Bacteriol. 178:1401-1411, 1996).
Clearly, there is a need for factors, such as spo-rel of the invention, that may be used to screen compounds for antibiotic activity and which may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is a need, therefore, for identification and characterization of such factors which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
The polypeptide of the present invention has amino acid sequence homology to known guanosine 3xe2x80x2, 5xe2x80x2-bis(diphosphate) synthetase/hydrolase protein.
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel spo-rel peptides by homology between the amino acid sequence set out in FIG. 2 [SEQ ID NO:2] and known amino acid sequences of other proteins such as Streptococcus equisimilis rel protein protein.
It is a further object of the invention, moreover, to provide polynucleotides that encode spo-rel polypeptides, particularly polynucleotides that encode the polypeptide herein designated spo-rel.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding spo-rel polypeptides in the sequence set out in FIG. 1 [SEQ ID NO:1], or a fragment, analogue or derivative thereof.
In another particularly preferred embodiment of the present invention there is a novel guanosine 3xe2x80x2, 5xe2x80x2-bis(diphosphate) synthetase/hydrolase protein from Staphylococcus aureus comprising the amino acid sequence of FIG. 2 [SEQ ID NO:2], or a fragment, analogue or derivative thereof.
In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the bacterial strain Staphylococcus aureus WCUH 29 contained in NCIMB Deposit No. 40771.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding spo-rel, particularly Staphylococcus spo-rel, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives, and compositions comprising same.
In accordance with another aspect of the present invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of spo-rel and polypeptides encoded thereby.
In accordance with this aspect of the invention there are provided novel polypeptides of Staphylococcus referred to herein as spo-rel as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing, and compositions comprising same.
Among the particularly preferred embodiments of this aspect of the invention are variants of spo-rel polypeptide encoded by naturally occurring alleles of the spo-rel gene.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned spo-rel polypeptides.
In accordance with yet another aspect of the present invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia: assessing spo-rel expression; to treat diseases and infections of the upper respiratory tract, e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory, e.g. empyema, lung abscess, cardiac, e.g. infective endocarditis, gastrointestinal, e.g. secretory diarrhea, splenic abscess, retroperitoneal abscess, CNS, e.g. cerebral abscess, eye, e.g. blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis, kidney and urinary tract, e.g. epididymitis, intrarenal and perinephric abscess, toxic shock syndrome, skin, e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis, and bone and joint, e.g. septic arthritis, osteomyelitis; assaying genetic variation; and administering a spo-rel polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a bacteria of the genus Staphylococcus.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to spo-rel polynucleotide sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against spo-rel polypeptides.
In accordance with another aspect of the present invention, there are provided spo-rel agonists which are also preferably bacteriostatic or bacteriocidal.
In accordance with yet another aspect of the present invention, there are provided spo-rel antagonists which are also preferably bacteriostatic or bacteriocidal.
In a further aspect of the invention there are provided compositions comprising a spo-rel polynucleotide or a spo-rel polypeptide for administration to a cell or to a multicellular organism.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.